Identification of the viral RNA promoter stem loop A (SLA)-binding site on Zika virus polymerase NS5

Wlodzimierz Bujalowski,Kyung H Choi,Paul J Bujalowski

doi:10.1038/s41598-020-70094-y

Wlodzimierz Bujalowski, Kyung H Choi + Show 1 more

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https://doi.org/10.1038/s41598-020-70094-y

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Abstract

Zika virus has recently emerged as an important human pathogen that has spread to more than 60 countries. Infection of a pregnant woman with Zika virus can cause severe brain malformations in the child such as microcephaly and other birth defects. Despite the medical importance of Zika virus infection, the mechanism of viral replication, a process commonly targeted by antiviral therapeutics, is not well understood. Stem-loop A (SLA), located in the 5′ untranslated region of the viral genome, acts as a promotor for viral replication and thus is critical for recognition of the viral genome by the viral polymerase NS5. However, how NS5 engages SLA is not clear. We have quantitatively examined the intrinsic affinities between Zika virus SLA and NS5, and identified the SLA-binding site on NS5. Amino acid substitutions in the thumb subdomain of the RNA-dependent RNA polymerase (RdRp) and the methyltransferase (MTase) domain reduced SLA-binding affinity, indicating that they each are part of the SLA-binding site. Furthermore, stopped-flow kinetic analysis of Zika NS5-, RdRp- and MTase–SLA interactions identified distinct intermediates during NS5 and SLA complex formation. These data suggest a model for SLA recognition and the initiation of flaviviral replication by NS5.

Highlights

Zika virus (ZIKV) is a member of the family Flaviviridae, and since 2007 has emerged as a major threat to global health, causing a series of epidemics in Micronesia, the South Pacific and both A mericas[1]
In the case of flaviviruses, the Stem-loop A (SLA) at the 5′ end of viral RNA plays a critical role by functioning as a promoter of the replication p rocess[10,11,12]
In order to understand how ZIKV NS5 recognizes the SLA promotor for initiation of RNA synthesis, we examined the roles that each of the NS5 domains play in binding to SLA

Summary

Introduction

Zika virus (ZIKV) is a member of the family Flaviviridae, and since 2007 has emerged as a major threat to global health, causing a series of epidemics in Micronesia, the South Pacific and both A mericas[1]. The smaller N-terminal domain functions as a methyltransferase (MTase), whose activity is necessary for the 5′-RNA cap formation and methylation that are important for viral RNA recognition by the host cell translational a pparatus[22,23]. The 5′-UTR of the viral genome includes a structured stem-loop, called stem-loop A (SLA, nt 1- 70) (Fig. 1a), which functions as a promoter and recruits the viral polymerase NS5 to initiate RNA synthesis at the 3′-end of the g enome[10,11,12,25,26]. We have examined the binding affinities of ZIKV SLA with the full-length NS5 and its two individual RdRp and MTase domains, and identified the location of the SLA-binding site on the surface of NS5. We further determined the kinetics of Zika NS5-, RdRp- and MTase–SLA complex formation, and observed the presence of intermediates in the NS5–SLA complex reaction

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